Typical procedures for analyzing biological materials, such as nucleic acid, protein, lipid, carbohydrate, and other biological molecules, involve a variety of operations starting from raw material. These operations may include various degrees of cell separation or purification, cell lysis, amplification or purification, and analysis of the resulting amplification or purification product.
As an example, in DNA-based blood analyses samples are often purified by filtration, centrifugation or by electrophoresis so as to eliminate all the non-nucleated cells, which are generally not useful for DNA analysis. Then, the remaining white blood cells are broken up or lysed using chemical, thermal or biochemical means in order to liberate the DNA to be analyzed. Next, the DNA is denatured by thermal, biochemical or chemical processes and amplified by an amplification reaction, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), and the like. The amplification step allows the operator to avoid purification of the DNA being studied because the amplified product greatly exceeds the starting DNA in the sample.
If RNA is to be analyzed the procedures are similar, but more emphasis is placed on purification or other means to protect the labile RNA molecule. RNA is usually copied into DNA (cDNA) and then the analysis proceeds as described for DNA.
Finally, the amplification product undergoes some type of analysis, usually based on sequence or size or some combination thereof In an analysis by hybridization, for example, the amplified DNA is passed over a plurality of detectors made up of individual oligonucleotide detector fragments that are anchored, for example, on electrodes. If the amplified DNA strands are complementary to the oligonucleotide detectors or probes, stable bonds will be formed between them (hybridization). The hybridized detectors can be read by observation using a wide variety of means, including optical, electromagnetic, electromechanical or thermal means.
Other biological molecules are analyzed in a similar way, but typically molecule purification is substituted for amplification, and detection methods vary according to the molecule being detected. For example, a common diagnostic involves the detection of a specific protein by binding to its antibody. Such analysis requires various degrees of cell separation, lysis, purification and product analysis by antibody binding, which itself can be detected in a number of ways. Lipids, carbohydrates, drugs and small molecules from biological fluids are processed in similar ways. However, we have simplified the discussion herein by focusing on nucleic acid analysis, in particular DNA analysis, as an example of a biological molecule that can be analyzed using the devices of the invention.
Some devices integrate more than one process step. For example, some devices are designed to accept biological samples previously prepared and to perform amplification and detection processes (that may involve denaturation, hybridization of target probes and reading of the probes).
In most cases, individual operations are carried out in separate chambers. So, there is a need to transfer partially processed samples from one chamber to another and, to this end, microfluidic connections are provided between subsequent chambers.
However, handling small volumes of liquids, such as required for biochemical analyses (in the range of few microliters) can be difficult, especially when capillary forces are involved. In fact, air bubbles may easily be encapsulated in a chamber when a sample is loaded into a microreactor. Essentially, the geometry of the chambers and the affinity of the sample with the material which the chambers are made of may produce very instable menisci when the chambers are filled with a liquid. The edges of the menisci may join in certain conditions and air bubbles can be entrapped within the liquid.
A single air bubble may occupy a relatively large fraction of the chamber, in view of its small volume (some microliters) and cause leakage toward another chamber through the microfluidic connections. In other words, a volume of the sample may be pushed away by the air bubble and may escape from the reaction chamber through the microfluidic connections. The analysis may be compromised, because important process parameters, such as volume, balance of reagents, pressure, temperature, are affected by the bubble. In any case, a lower amount of sample is available for processing.
The object of the invention is to provide chemical microreactor and a method for carrying out a chemical reaction that are free from the above described limitations.